WO2024050860A1 - 一种乳腺叶状肿瘤的诊断试剂及其应用 - Google Patents

一种乳腺叶状肿瘤的诊断试剂及其应用 Download PDF

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WO2024050860A1
WO2024050860A1 PCT/CN2022/118894 CN2022118894W WO2024050860A1 WO 2024050860 A1 WO2024050860 A1 WO 2024050860A1 CN 2022118894 W CN2022118894 W CN 2022118894W WO 2024050860 A1 WO2024050860 A1 WO 2024050860A1
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runx2
breast
phyllodes
malignant
detection
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French (fr)
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聂燕
宋尔卫
雷蓉
黄红颜
贺诗施
艾力菲热伊力哈木
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中山大学孙逸仙纪念医院
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  • the invention relates to the field of medical biotechnology, and in particular to a diagnostic reagent for breast phyllodes tumors and its application.
  • Phyllodes tumor is a rare fibroepithelial tumor, accounting for 0.3% to 1% of all breast tumors.
  • the commonly used clinical preoperative diagnosis of breast phyllodes tumors includes breast ultrasound, mammography, breast MRI, preoperative fine needle aspiration and core needle aspiration, etc.
  • the preoperative diagnosis rate of phyllodes tumors is very low. It is only about 76%, and postoperative histopathological examination is the gold standard for diagnosis.
  • the histopathological diagnosis of phyllodes tumors is somewhat subjective and insufficient to predict the clinical prognosis of individual patients.
  • borderline and malignant phyllodes tumors not only grow rapidly, but are also prone to local recurrence and distant metastasis.
  • Blood metastasis is the most common way of metastasis, and lung and bone are the most common metastasis sites.
  • the recurrence rate of malignant phyllodes tumors of the breast is as high as 53.1%, and the metastasis rate is as high as 43.1%. Once recurrence and metastasis occur, the patient will die within a short period of time.
  • the mortality rate of malignant phyllodes tumors is approximately 16.3%.
  • the treatment of breast phyllodes tumors is mainly surgical treatment.
  • RUNX2 is an important member of the RUNX family of transcription factors and is named because it contains a runt domain.
  • the biological role of RUNX2 is mainly as a specific transcription factor for osteogenic differentiation, regulating the transcription of genes such as type I collagen, osteomodulin, osteocalcin, col1a1, col1a2, bone sialoprotein (BSP) and fibronectin. It plays an important role in the formation and differentiation of osteoblasts, differentiation and maturation of chondrocytes, the formation and resorption of osteoclasts, and the synthesis of bone matrix proteins.
  • BSP bone sialoprotein
  • RUNX2 is related to the biological behavior of malignant phyllodes tumors of the breast.
  • the present invention discloses the technical application of RUNX2 as a biological behavioral marker of malignant phyllodes tumors of the breast.
  • the mRNA expression level of RUNX2 in malignant phyllodes tumor tissues is approximately 25 times that of benign phyllodes tumor tissues. As the number of tumor recurrences increases, the expression of RUNX2 in tissues also increases simultaneously.
  • the expression of RUNX2 mRNA in malignant breast tumors has a certain positive correlation with the proliferation, migration and invasion abilities of malignant breast phyllodes tumor cells. Prove that RUNX2 can be used as a marker for the diagnosis or detection of malignant phyllodes tumors of the breast. According to the expression levels of RUNX2 in breast phyllodes tumors of different grades, assisted with conventional tissue biopsy, diagnosis, differential diagnosis, grading and prognosis analysis of breast phyllodes tumors can be performed.
  • RUNX2 as a marker in preparing diagnostic or detection reagents (including kits) for malignant phyllodes tumors of the breast.
  • the application includes: the use of RUNX2 as a marker in the preparation of detection reagents for early screening or prognosis prediction of breast malignant phyllodes tumors.
  • the diagnostic or detection reagent includes specific RUNX2 primers or RUNX2 antibodies.
  • the method of diagnosis or detection can be: detecting the RUNX2 expression level of malignant breast phyllodes tumor specimens in various biological states through qRT-PCR or immunohistochemistry, establishing a detection model, and obtaining standard data for diagnosis or detection. According to breast malignancy The RUNX2 expression level of the phyllodes tumor specimens to be tested was judged.
  • qRT-PCR or immunohistochemistry can be used to detect the RUNX2 expression level of breast malignant phyllodes tumor specimens, and the biology of breast malignant phyllodes tumor specimens can be analyzed based on the RUNX2 expression level. judge the situation.
  • the sequence of the preferred specific primer for RUNX2 is:
  • RUNX2 The preferred antibodies for RUNX2 are: MBL Anti-Runx2#D130-3, Abcam recombinant Anti-RUNX1/AML1+RUNX3+RUNX2 antibody [EPR3099]#ab92336.
  • the present invention has the following beneficial effects:
  • the present invention discloses for the first time that the overexpression of RUNX2 in breast malignant phyllodes tumor specimens is significantly related to the biological behavior of breast malignant phyllodes tumors, and can be used as a marker for diagnosing and detecting breast malignant phyllodes tumors and screening anti-breast phyllodes tumors drugs. , which has high clinical application value in early screening diagnosis, prognosis prediction and screening of targeted anti-tumor drugs.
  • the test results of the present invention prove that RUNX2 is lowly expressed in benign phyllodes tumor tissues and highly expressed in malignant phyllodes tumor tissues.
  • the expression level of RUNX2 mRNA in malignant tumors is approximately 25 times that of benign phyllodes tumor tissues. The difference is very obvious, proving that RUNX2 has strong specificity as a marker, and provides strong theoretical support for the feasibility and accuracy of the RUNX2 marker-related diagnostic detection reagent or kit of the present invention.
  • the RUNX2 detection primer sequence and detection antibody of the present invention can be used as important components of the RUNX2 detection kit, thereby making the detection of malignant phyllodes tumors of the breast simpler, faster and more accurate, and have good clinical application value and contribute to To further improve the diagnostic level of malignant phyllodes tumors of the breast, it is expected to become the first diagnostic test kit for malignant phyllodes tumors of the breast.
  • Figure 1 A is a histogram of RUNX2 mRNA expression levels in benign and malignant breast phyllodes tumor tissues; B: is a protein immunoblot gel imaging image of RUNX2 protein expression test in benign and malignant breast phyllodes tumor tissues; C: This is a graphic representation of the RUNX2 expression levels in paraffin sections of benign and malignant phyllodes tumors and the RUNX2 expression levels in paraffin sections of multiple recurrences of tumor tissue from the same patient.
  • Figure 2 A is a graphical representation of the test results of knocking down the RUNX2 gene of the breast malignant phyllodes tumor cell line SYSH-MPT-01 (i.e., the cell line HJP-0320, disclosed in the patent application with publication number CN111019898A) and changing the cell proliferation ability.
  • SYSH-MPT-01 i.e., the cell line HJP-0320, disclosed in the patent application with publication number CN111019898A
  • B knocks down the RUNX2 gene of the breast malignant phyllodes tumor cell line SYSH-MPT-01, and the results of the test on changes in cell clonogenic ability
  • C shows the knockdown of the RUNX2 gene of the breast malignant phyllodes tumor cell line SYSH-MPT-01, Graphical representation of cell cycle change test results
  • D is a graphic representation of the knockdown of the RUNX2 gene in the breast malignant phyllodes tumor cell line SYSH-MPT-01, and changes in cell migration and invasion capabilities
  • E is a graphic representation of the knockdown of the breast malignant phyllodes tumor cell line Illustration of the test results of the RUNX2 gene of SYSH-MPT-01 and the changes in cell collagen contraction ability.
  • FIG. 3 A is a graphical representation of the test results of changes in cell proliferation ability overexpressing RUNX2 in the benign breast phyllodes tumor cell line SYSH-BPT-01 (i.e., the cell line GLK-1010, disclosed in the patent application with publication number CN111019897A) ; B is a graphical representation of the test results of the overexpression of RUNX2 in the benign breast phyllodes tumor cell line SYSH-BPT-01, and the changes in the clonogenic ability of the cells; C is the overexpression of RUNX2 in the benign breast phyllodes tumor cell line SYSH-BPT-01 RUNX2, a graphical representation of the test results of cell cycle changes; D is a graphical representation of the test results of overexpression of RUNX2 in the benign breast phyllodes tumor cell line SYSH-BPT-01, and the changes in cell migration and invasion capabilities; E is a graphic representation of the test results of the benign breast phyllodes tumor RUNX2 is overex
  • Figure 4 Illustration of the results of a trial on the impact of RUNX2 on the prognosis of patients with breast phyllodes tumors.
  • Figure 5 Illustration of the test results of knockdown of RUNX2 in the malignant phyllodes tumor cell line SYSH-MPT-01 on subcutaneous tumor formation ability and tumor growth; A is the change in tumor volume at the corresponding time point, and B is the picture of the tumors in each group .
  • Example 1 Analysis of RUNX 2 expression profile of breast phyllodes tumors
  • RUNX2-specific primers to perform fluorescence quantitative PCR to detect the expression levels of RUNX2 mRNA in different types of breast phyllodes tumor tissues.
  • Reverse transcription to synthesize cDNA Reverse transcription to synthesize cDNA: Add 1 ⁇ g of template RNA and 4 ⁇ l of reverse transcriptase SuperScript II mix (containing Buffer, dNTP, HiScript II reverse transcriptase, RNase, Random primers/Oligo dT) in the PCR tube.
  • Real-time quantitative PCR amplification Dilute the template cDNA 3 times and mix well for later use. Each experimental group was set up with 3 parallel tubes. Reaction system: 1 ⁇ l cDNA, 5 ⁇ l SYBR green dye, 0.3 ⁇ l forward primer, 0.3 ⁇ l reverse primer, 3.4 ⁇ l ddH 2 O, centrifuge and mix. Reaction conditions: 95°C for 5min, 95°C for 30 seconds, 55°C-60°C (depending on the annealing temperature) for 30s, a total of 40 cycles. GAPDH was used as an internal reference to analyze the relative transcription levels of genes. The primer sequences are as follows:
  • Example 2 Effects of knocking down RUNX2 on proliferation, migration, invasion, cell cycle and collagen contraction ability of malignant phyllodes tumor cells
  • the shRNA sequence is as follows:
  • the present invention identifies RUNX2 as a specific molecular marker for malignant phyllodes tumors of the breast.
  • This marker is highly expressed in malignant phyllodes tumor cells of the breast and promotes the progression of malignant phyllodes tumors of the breast. Knocking down the expression of RUNX2 can inhibit the proliferation, migration, and invasion of malignant phyllodes tumors of the breast.
  • RUNX2 has significant clinical application value in the development of auxiliary diagnosis of breast phyllodes tumors.

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Abstract

涉及医药生物技术领域，尤其是RUNX2作为标志物在制备乳腺恶性叶状肿瘤诊断或检测试剂中的应用。检测试剂可根据RUNX2在不同分级乳腺叶状肿瘤中的表达水平，辅助常规组织活检，可以对乳腺叶状肿瘤进行诊断、鉴别诊断及分级和预后分析。RUNX2检测引物序列和检测抗体是诊断或检测试剂或试剂盒的重要组分，从而使得乳腺恶性叶状肿瘤的检测更为简便快捷准确，具有很好的临床应用价值，有助于进一步提高乳腺恶性叶状肿瘤的诊断水平，有望成为首个针对乳腺恶性叶状肿瘤的诊断检测试剂盒。

Description

一种乳腺叶状肿瘤的诊断试剂及其应用 技术领域
本发明涉及医药生物技术领域，具体涉及一种乳腺叶状肿瘤的诊断试剂及其应用。
背景技术
乳腺叶状肿瘤(Phyllodes tumor,PT)是一种罕见的纤维上皮性肿瘤，占所有乳腺肿瘤的0.3％至1％。目前临床常用的乳腺叶状肿瘤术前诊断包括乳腺彩超、钼靶、乳腺核磁共振、术前肿块细针穿刺及空芯针穿刺等，但综合所有的诊断方法，叶状肿瘤的术前确诊率也仅为76％左右，术后病理组织学检查才是诊断的金标准。同时由于个体差异，缺乏明确的标准及临界值，基于形态学确诊叶状肿瘤仍具有挑战性。因此，叶状肿瘤的病理组织学诊断具有一定主观性，不足以预测个体患者的临床预后。
根据现有的文献研究，交界性、恶性叶状肿瘤不仅生长快，而且易局部复发和远处转移，血液转移是最常见的转移方式，肺、骨是最常见的转移部位。乳腺恶性叶状肿瘤的复发率高达53.1％，转移率高达43.1％。一旦出现复发、转移，患者会在短期内死亡，恶性叶状肿瘤的死亡率约为16.3％。乳腺叶状肿瘤的治疗主要是手术治疗，恶性叶状肿瘤由于其具有高复发率、高转移率等特点，其临床预后差。不同于乳腺癌，乳腺恶性叶状肿瘤术后的辅助治疗手段如化疗、放疗等对其治疗效果均不佳，靶向治疗及免疫治疗目前也无研究报道具有临床获益。且由于乳腺叶状肿瘤较为罕见，缺乏大规模的临床数据与基础研究，明确其诊断的特异性分子标志物及其相关的诊断试剂及相应药物的研发更为匮乏。故亟需寻找乳腺叶状肿瘤的特异性分子标志物，对于提高术前诊断率、改善患者预后具有十分重要的作用。
RUNX2是转录因子RUNX家族的重要成员，其因含有runt结构域而被命名。RUNX2的生物学作用主要是作为成骨分化的特异性转录因子，调控一型胶原、骨调素、骨钙蛋白、col1a1、col1a2、骨涎蛋白(BSP)和纤维连接蛋白等基因的转录，在成骨细胞形成和分化、软骨细胞分化和成熟、破骨细胞的形成和吸收及骨基质蛋白的合成中起到重要作用。但未见有RUNX2与乳腺恶性叶状肿瘤生物学行 为相关的报道。
发明内容
为克服以上技术问题，本发明公开了RUNX2作为乳腺恶性叶状肿瘤生物学行为标志物相关的技术应用。
发明人在临床样本和体外细胞实验中发现，RUNX2在良性叶状肿瘤组织中低表达，在恶性叶状肿瘤组织高表达，恶性中RUNX2的mRNA表达水平约为良性叶状肿瘤组织的25倍。随着肿瘤复发次数的增加，RUNX2在组织中的表达量也同步增加。恶性中RUNX2mRNA的表达量与乳腺恶性叶状肿瘤细胞的增殖、迁移和侵袭能力呈一定地正相关关系。证明RUNX2可作为诊断或检测乳腺恶性叶状肿瘤的标志物。根据RUNX2在不同分级乳腺叶状肿瘤中的表达水平，辅助常规组织活检，可以对乳腺叶状肿瘤进行诊断、鉴别诊断及分级和预后分析。
由此公开了以下应用:
RUNX2作为标志物在制备乳腺恶性叶状肿瘤诊断或检测试剂(包括试剂盒)中的应用。
优选的，所述的应用包括：RUNX2作为标志物在制备乳腺恶性叶状肿瘤早期筛选或预后预测检测试剂中的应用。
优选的，所述诊断或检测试剂包括特异性的RUNX2引物或RUNX2抗体。
诊断或检测的方法可以是：通过qRT-PCR或免疫组化的方法检测各个生物学状态的乳腺恶性叶状肿瘤标本的RUNX2表达水平，建立检测模型，获得诊断或检测的标准数据，根据乳腺恶性叶状肿瘤待检标本的RUNX2表达水平进行判断。
所述诊断或检测试剂或试剂盒的使用方法：可用qRT-PCR或免疫组化的方法检测乳腺恶性叶状肿瘤标本的RUNX2表达水平，根据RUNX2表达水平情况对乳腺恶性叶状肿瘤标本的生物学状况进行判断。
RUNX2优选的特异性引物的序列为：
Forward Primer：5’-CGCCTCACAAACAACCACAG-3’；(序列表SEQ ID NO.1)
Reverse Primer：5’-TCACTGTGCTGAAGAGGCTG-3’；(序列表SEQ ID NO.2)
RUNX2优选抗体为：MBL Anti-Runx2#D130-3,Abcam重组Anti-RUNX1/AML1+RUNX3+RUNX2抗体[EPR3099]#ab92336。
与现有技术相比，本发明具有如下的有益效果：
1.本发明首次公开乳腺恶性叶状肿瘤标本RUNX2的过表达与乳腺恶性叶状肿瘤的生物学行为显著相关，可作为诊断检测乳腺恶性叶状肿瘤及筛选抗乳腺恶性叶状肿瘤药物的标志物，在早期筛查诊断、预后预测及筛选靶向抗肿瘤药物等方面均具有较高临床应用价值。
2.本发明试验结果证明，RUNX2在良性叶状肿瘤组织中低表达，在恶性叶状肿瘤组织高表达，恶性中RUNX2mRNA的表达水平约为良性叶状肿瘤组织的25倍。差异量非常明显，证明RUNX2作为标志物的特异性强，为本发明的RUNX2标志物相关诊断检测试剂或试剂盒的可行性和准确性提供了有力的理论支撑。
3.本发明的RUNX2检测引物序列和检测抗体，可用于RUNX2检测试剂盒的重要组分，从而使得乳腺恶性叶状肿瘤的检测更为简便快捷准确，具有很好的临床应用价值，有助于进一步提高乳腺恶性叶状肿瘤的诊断水平，可望成为首个针对乳腺恶性叶状肿瘤的诊断检测试剂盒。
附图说明
图1:A是良性和恶性乳腺叶状肿瘤组织中RUNX2的mRNA表达水平的柱状图；B：是良性和恶性乳腺叶状肿瘤组织中RUNX2蛋白表达试验的蛋白免疫印迹凝胶成像图；C：是良、恶性叶状肿瘤石蜡切片中的RUNX2表达水平及同一患者多次复发的肿瘤组织石蜡切片中RUNX2表达水平情况图示。
图2：A是敲降乳腺恶性叶状肿瘤细胞系SYSH-MPT-01(即细胞系HJP-0320,公开于公布号为CN111019898A的专利申请中)的RUNX2基因，细胞增殖能力改变试验结果图示；B敲降乳腺恶性叶状肿瘤细胞系SYSH-MPT-01的RUNX2基因，细胞克隆形成能力改变试验结果图示；C是敲降乳腺恶性叶状肿瘤细胞系SYSH-MPT-01的RUNX2基因，细胞周期改变试验结果图示；D是敲降乳腺恶性叶状肿瘤细胞系SYSH-MPT-01的RUNX2基因，细胞迁移和侵袭能力改变试验结果图示；E是敲降乳腺恶性叶状肿瘤细胞系SYSH-MPT-01的RUNX2基因，细胞胶原收缩能力改变试验结果图示。
图3：A是在乳腺良性叶状肿瘤细胞系SYSH-BPT-01(即细胞系GLK-1010,公开于公布号为CN111019897A的专利申请中)中过表达RUNX2，细胞增殖能力改变试验结果图示；B是在乳腺良性叶状肿瘤细胞系SYSH-BPT-01中过表达RUNX2，细胞的克隆形成能力改变试验结果图示；C是在乳腺良性叶状肿瘤细胞系SYSH-BPT-01中过表达RUNX2，细胞周期的改变试验结果图示；D是在乳腺良性 叶状肿瘤细胞系SYSH-BPT-01中过表达RUNX2，细胞迁移及侵袭能力改变试验结果图示；E是在乳腺良性叶状肿瘤细胞系SYSH-BPT-01中过表达RUNX2，细胞胶原收缩能力改变试验结果图示。
图4：RUNX2对乳腺叶状肿瘤患者预后影响试验结果图示。
图5：敲降恶性叶状肿瘤细胞系SYSH-MPT-01的RUNX2，对皮下成瘤能力及肿瘤生长影响试验结果图示；A是相应时间点肿瘤体积的变化情况,B是各组肿瘤图片。
具体实施方式
下面结合实施例对本发明作进一步的说明。
实施例1：乳腺叶状肿瘤RUNX 2表达谱分析
1.采用RUNX2特异性引物，进行荧光定量PCR，检测不同类型乳腺叶状肿瘤组织中RUNX2mRNA表达水平。
(1)分别选取3例良性叶状肿瘤组织标本、10例恶性叶状肿瘤组织标本。按以下步骤进行实验：将组织用冷冻研磨机研碎，加入Trizol裂解液，用移液器将裂解物转移至1.5mlEP管中，反复吹打或振荡以裂解细胞。室温静置5min，按每1ml Trizol裂解液加入0.2ml氯仿，用力振荡15s，室温静置2-3min后，12000x g 4℃离心15min，取上层水相至新EP管中，加入等体积异丙醇沉淀RNA；12000x g 4℃离心10min；加入75％乙醇清洗，7500x g 4℃离心5min，弃上清；室温下超静台内晾干RNA沉淀，溶解于适量Rnase-free水中，测定RNA浓度及纯度。反转录合成cDNA：反转录合成cDNA：在PCR管中加入模板RNA 1μg，4μl反转录酶SuperScript II mix(含Buffer、dNTP、HiScript II逆转录酶、RNA酶、Random primers/Oligo dT)，补充RNase-free水至20μl，65℃5min，冰上放置5min后加入5×buffer 8μl及0.1M DTT 2μl，混匀后补加ddH2O至40μl，反应条件：50℃15min，85℃15s，4℃保存。
实时定量PCR扩增：将模板cDNA稀释3倍并混匀备用。每个实验组设置3个平行管。反应体系：cDNA 1μl，SYBR green染料5μl，正向引物0.3μl，反向引物0.3μl，ddH 2O 3.4μl，离心混匀。反应条件：95℃5min，95℃30秒，55℃-60℃(视退火温度而定)30s，共40个循环。以GAPDH作为内参分析检测基因的相对转录水平。引物序列如下：
RUNX2Forward：5’-CGCCTCACAAACAACCACAG-3’；(序列表SEQ ID NO.1)
RUNX2 Reverse：5’-TCACTGTGCTGAAGAGGCTG-3’。(序列表SEQ ID NO.2)
定量分析mRNA相对表达水平。统计数据根据三次重复实验结果计算平均值，显著性差异由T-检验来判断，以P<0.05定义为有统计学差异。
(2)实验结果：如图1(A)所示，RUNX2在良性叶状肿瘤组织中低表达，在恶性叶状肿瘤组织高表达，恶性中RUNX2mRNA的表达水平约为良性叶状肿瘤组织的25倍。
2.采用蛋白免疫印迹(Western blot)实验检测不同类型叶状肿瘤组织中RUNX2蛋白表达水平。
(1)实验方法：SDS-PAGE电泳、转膜、封闭之后，采用RUNX2特异性抗体作为一抗孵育，进一步采用辣根过氧化物酶标二抗孵育之后进行凝胶成像分析。
(2)实验结果:如图1(B)显示，10例恶性叶状肿瘤组织中RUNX2蛋白表达量较高，而在3例良性叶状肿瘤组织中RUNX2蛋白不表达或低表达。
3.使用RUNX2的特异性抗体，进行免疫组织化学染色，评估良、乳腺恶性叶状肿瘤组织中RUNX2蛋白水平。
(1)实验方法：实验所采用病理切片均来自于临床确诊病例。按以下步骤进行石蜡包埋组织免疫组化染色：60℃烘烤处理，入预热二甲苯脱蜡两次，每次5min；脱蜡后的切片经100％-95％-80％-70％-50％乙醇梯度及蒸馏水水化，每个梯度放置5min；0.01M(pH6.0)柠檬酸缓冲液高压热修复10min，自然冷却后PBS洗三次，每次5min；入0.3％过氧化氢溶液处理30min，消除内源性过氧化物酶活性；PBS洗三次，每次5min；10％羊血清37℃封闭1h；滴加羊血清稀释一抗工作液(AA4)，4℃孵育过夜，PBS清洗；加入生物素标记二抗工作液，37℃孵育20min，PBS清洗三次；加入辣根过氧化物酶标记链霉亲和素，37℃孵育20min，PBS清洗三次；滴加DAB显色液，室温避光显色2min，PBS洗去多余显色液；苏木素复染，蒸馏水清洗；50％-70％-80％-90％-100％-100％乙醇梯度逐级脱水，每个梯度5min；中性树脂封片；显微成像系统拍片。
(2)实验结果:如图1(C)所示：RUNX2在乳腺恶性叶状肿瘤细胞中高表达，在良性中低表达(C)。随着肿瘤复发次数的增加，RUNX2在组织中的表达量也同步增加(C)。
实施例2：敲低RUNX2对恶性叶状肿瘤细胞增殖、迁移、侵袭、细胞周期及 胶原收缩能力的影响
1.敲低RUNX2对恶性叶状肿瘤细胞增殖的影响
(1)实验方法：接种恶性叶状肿瘤细胞于96孔板和6孔板中，采用1ipo3000瞬时转染shRNA，至乳腺恶性叶状肿瘤细胞中，敲低RUNX2的表达接种敲低RUNX2表达及未进行基因敲低的对照组细胞，采用CCK8法，测试不同时间点(1-4天)细胞存活率，绘制细胞增殖曲线。接种敲低RUNX2表达及未进行基因敲低的对照组细胞至60mm中皿的完全培养基中，每皿200个细胞，培养14天后，计数克隆形成数量。
shRNA序列如下：
shRUNX2 #1:GGACGAGGCAAGAGTTTCA；(序列表SEQ ID NO.3)
shRUNX2 #2:CCAAATTTGCCTAACCAGA。(序列表SEQ ID NO.4)
(2)实验结果:如图2(A-B)所示。
2.敲低RUNX2对恶性叶状肿瘤细胞周期的影响
(1)实验方法：接种敲低表达及对照细胞，培养24h后，收集细胞，用预冷的PBS洗细胞2-3次，离心(1500rpm，4min)弃掉上清，在沉淀中加入少量PBS，重悬细胞，再将重悬细胞加入到4℃预冷的70％冰乙醇中固定，封口膜封口，4℃过夜。PBS洗两次离心去上清液(2000rpm 4min)。加入100ul 100ug/ml RNase A和0.2％Triton X-100重悬细胞。加入400ul 50ug/ml PI，涡旋振荡混匀，室温避光孵育30min。流式细胞仪检测细胞周期，一般计数10万个细胞，在激发波长488nm波长处检测红色荧光，而后用FlowJo软件分析细胞周期时相分布，分析时，使用FL2-w和FL2-A显示，去除粘连在一起的细胞。
(2)实验结果：如图2(C)敲低RUNX2后的细胞阻滞在G1期。
3.敲低RUNX2对恶性叶状肿瘤细胞迁移、侵袭能力的影响
(1)实验方法：将敲低RUNX2表达及对照组细胞以2x10 4/孔的密度接种于Transwell板(Costar)上层小室无血清培养基中或预铺Matrigel薄层上层培养室中，下层为含16％胎牛血清的完全培养基，每组设置三个重复孔；37℃培养箱分别培养8小时或24小时后，用4％多聚甲醛固定15min，小心擦去膜上层细胞，利用0.5％结晶紫对下室细胞进行染色，显微镜下计数发生迁移或侵袭的细胞数目。
(2)实验结果；如图2(D)显示，RUNX2敲低表达组细胞迁移及侵袭能力显著下降。
4.敲低RUNX2对恶性叶状肿瘤胶原收缩能力的影响
(1)实验方法：用含乙酸和NaOH的DMEM培养基稀释Ⅰ型鼠尾胶原，叶状肿瘤细胞悬液与稀释后的鼠尾胶原1:1混合后，接种到24孔板中，加入含血清的培养基4小时后，换上无血清DMEM培养基孵育过夜，使胶与孔壁分离，8小时后观察、测量收缩后的直径。
(2)实验结果如图2(E)所示。
实施例3.过表达RUNX2对叶状肿瘤细胞增殖、迁移、侵袭、细胞周期及胶原收缩能力的影响
1.过表达RUNX2对良性乳腺叶状肿瘤细胞增殖的影响
(1)实验方法:接种原代良性叶状肿瘤细胞至96孔板和6孔板中，使用转染试剂lipo3000和p3000转染RUNX2过表达质粒(4μg/孔)，转染4-6小时后更换正常完全培养基，收集细胞提取RNA样品，检测RUNX2过表达水平。接种过表达RUNX2及未进行处理的对照组细胞，采用CCK8法，测试不同时间点(1-4天)细胞存活率，绘制细胞增殖曲线。接种过表达RUNX2及未进行处理的对照组细胞至60mm中皿完全培养基中，每皿200个细胞，培养14天后，计数克隆形成数量。
(2)实验结果如图3(A-B)所示。
2.过表达RUNX2对良性叶状肿瘤细胞周期的影响
(1)实验方法：接种过表达及对照细胞，培养24h后，收集细胞，用预冷的PBS洗细胞2-3次，离心(1500rpm，4min)弃掉上清，在沉淀中加入少量PBS，重悬细胞，再将重悬细胞加入到4℃预冷的70％冰乙醇中固定，封口膜封口，4℃过夜。PBS洗两次离心去上清液(2000rpm 4min)。加入100ul 100ug/ml RNase A和0.2％Triton X-100重悬细胞。加入400ul 50ug/ml PI，涡旋振荡混匀，室温避光孵育30min。流式细胞仪检测细胞周期，一般计数10万个细胞，在激发波长488nm波长处检测红色荧光，而后用FlowJo软件分析细胞周期时相分布，分析时，使用FL2-w和FL2-A显示，去除粘连在一起的细胞。
(2)实验结果：如图3(C)过表达RUNX2后的细胞活跃在S期。
3.过表达RUNX2对乳腺良性叶状肿瘤细胞迁移和侵袭能力的影响
(1)实验方法：将过表达RUNX2及未进行处理的对照组细胞以1*10 4/孔的密度接种于Transwell板(Costar)上层小室无血清培养基中或预铺Matrigel薄层上层培养室中，下层为含16％胎牛血清的完全培养基，每组设置三个重复孔；37℃培养箱分别培养8小时或24小时后，小心擦去膜上层细胞，利用结晶紫对下室细胞进行染色，显微镜下计数发生迁移或侵袭的细胞数目。
(2)实验结果如图3(D)所示。
4.过表达RUNX2对恶性叶状肿瘤胶原收缩能力的影响
(1)实验方法：用含乙酸和NaOH的DMEM培养基稀释Ⅰ型鼠尾胶原，叶状肿瘤细胞悬液与稀释后的鼠尾胶原1:1混合后，接种到24孔板中，加入含血清的培养基4小时后，换上无血清DMEM培养基孵育过夜，使胶与孔壁分离，8小时后观察、测量收缩后的直径。
(2)实验结果如图3(E)所示。
实施例4.RUNX2对乳腺叶状肿瘤患者预后的影响
1.RUNX2对乳腺叶状肿瘤患者预后的影响
(1)实验方法：取237例乳腺叶状肿瘤患者的新鲜冰冻切片，进行免疫组化染色，由两位病理科医生分别阅读切片，根据染色强度和染色阳性比例，分为RUNX2高表达组和RUNX2低表达组，评估RUNX2表达与总生存期OS的影响，无进展生存期DFS的影响。
(2)实验结果：如图4(A-B)所示，RUNX2高表达组的OS和DFS显著低于RUNX2低表达组。
实施例5敲低RUNX2基因影响小鼠体内肿瘤的生长
1.敲低RUNX2基因抑制小鼠皮下移植瘤成瘤及生长的影响
(1)实验方法：构建乳腺恶性叶状肿瘤皮下移植瘤模型：取恶性叶状肿瘤细胞、稳定敲降RUNX2的叶状肿瘤细胞，稀释至5x10 7/ml，取100μl混匀的细胞悬液，于对照组小鼠、实验组小鼠分别于左前肢腋窝部皮下乳房脂肪垫处接种，每4天测量一次肿瘤长径和短径，根据公式V＝1/2(L×W 2)计算移植瘤平均体积。待对照组肿瘤体积达到直径1.5cm时处死小鼠，并绘制肿瘤生长变化曲线。
(2)实验结果：如图5(A-B)所示，与对照组相比，接种稳定敲降RUNX2叶状肿瘤细胞小鼠的肿瘤成瘤及生长明显被抑制。
综上所述，本发明鉴定RUNX2作为乳腺恶性叶状肿瘤的特异性分子标志物，该标志物在乳腺恶性叶状肿瘤细胞中高表达，促进乳腺恶性叶状肿瘤的进展。敲低RUNX2的表达可以抑制乳腺恶性叶状肿瘤的增殖、迁移、侵袭。RUNX2作为乳腺恶性叶状肿瘤标志物，在乳腺叶状肿瘤辅助诊断开发中具有显著临床应用价值。

Claims (6)

  1. RUNX2作为标志物在制备乳腺恶性叶状肿瘤诊断或检测试剂中的应用。
  2. 如权利要求1所述的应用，其特征在于，所述的应用包括：
    (1)RUNX2作为标志物在制备乳腺恶性叶状肿瘤早期筛选检测试剂中的应用；
    (2)RUNX2作为标志物在制备乳腺恶性叶状肿瘤预后预测试剂中的应用。
  3. 如权利要求1或2所述的应用，其特征在于，所述诊断或检测试剂包括特异性RUNX2引物或RUNX2抗体。
  4. 如权利要求3所述的应用，其特征在于，所述诊断或检测试剂包括以下RUNX2引物，其序列为：
    Forward Primer：5’-CGCCTCACAAACAACCACAG-3’；
    Reverse Primer：5’-TCACTGTGCTGAAGAGGCTG-3’；
  5. 如权利要求3所述的应用，其特征在于，所述诊断或检测试剂包括以下RUNX2抗体：MBL Anti-Runx2#D130-3,Abcam重组Anti-RUNX1/AML1+RUNX3+RUNX2抗体[EPR3099]#ab92336。
  6. 如权利要求1所述的应用，其特征在于，所述诊断或检测试剂的使用方法为：用qRT-PCR或免疫组化的方法检测乳腺恶性叶状肿瘤标本的RUNX2表达水平。
PCT/CN2022/118894 2022-09-08 2022-09-15 一种乳腺叶状肿瘤的诊断试剂及其应用 WO2024050860A1 (zh)

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